Compositions for repair of defects in tissues, and methods of making the same

ABSTRACT

Tissue repair compositions, particularly bone repair compositions, containing (a) bone fragments and (b) homogenized connective tissue, and methods for making the same are provided. Some of the inventive tissue repair compositions contain a radioprotectant. The compositions can be used in the form of an injectable gel, an injectable paste, a paste, a putty, or a rehydratable freeze-dried form. Kits for using such tissue repair compositions are also provided.

RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.12/188,127, filed Aug. 7, 2008, and a continuation-in-part ofapplication Ser. No. 12/394,629, filed Feb. 27, 2009, which is acontinuation of application Ser. No. 11/247,230, filed Oct. 12, 2005,all of which are herein incorporated by reference in their entirety.

FIELD OF INVENTION

The present invention provides tissue repair compositions comprisinghomogenized connective tissue and bone fragments. The tissue repaircompositions may be used to repair and regenerate tissues.

BACKGROUND OF THE INVENTION

The ability to promote tissue regrowth in vivo can facilitate woundhealing and post-surgical recovery of patients who have suffered tissuedamage or destruction. A variety of methods and compositions have beenused to repair or regenerate bone tissue in vivo. The need for suchmethods and compositions is readily apparent, when considering that in1999, approximately 500,000 bone graft procedures were performed in theUnited States alone. Ideal bone graft materials for use in suchprocedures possess characteristics necessary to induce new bone growth,namely osteoconductivity and osteoinductivity.

Osteoconductivity refers to a graft's ability to support the attachmentof new osteoblasts and osteoprogenitor cells. The osteoconductivecomponents of a graft provide an interconnected structure through whichnew cells can migrate and new blood vessels can form. Osteoinductivityrefers to the ability of a graft to induce nondifferentiated stem cellsor osteoprogenitor cells to differentiate into osteoblasts.

In 1998, nine out of ten bone graft procedures performed in the UnitedStates involved the use of either autograft or allograft bone tissue.Despite the benefits of autografts and allografts, the limitations ofeach have necessitated the pursuit of alternative graft materials. Usingbasic criteria necessary to a successful graft (e.g., osteoconductionand osteoinduction), investigators have developed several bone graftsubstitutes. These can contain a variety of materials, including naturaland synthetic polymers, ceramics, and composites; and in some instances,production of bone graft substitutes can involve biotechnologicalstrategies (i.e., factor- and/or cell-based strategies).Non-demineralized bone, such as freeze-dried cortical particulate, isthe most often used osteoconductive bone grafting material.

Osteoinductive substances found in some bone graft substitutes aredemineralized bone particles and/or powder. Contained in theextracellular matrix of bone tissue is a full cocktail of bone growthfactors, proteins, and other bioactive materials necessary forosteoinduction and, ultimately, successful bone healing. To capitalizeon this cocktail of proteins, bone tissue can be demineralized, leavingthe osteoinductive agents in the demineralized bone matrix (DBM). Suchosteoinductive DBM can be incorporated into a number of different bonegraft substitutes.

While a number of different materials thought to enhanceosteoconductivity (i.e., purified or partially purified polymers) havebeen used in bone graft substitutes, new, more easily prepared,osteoconductive/structural materials for combining with demineralized ornon-demineralized bone to produce a bone graft substitute are desirable.

SUMMARY OF THE INVENTION

The present invention provides a carrier consisting of or consistingessentially of one or more homogenized connective tissues. An example ofa connective tissue that may be homogenized to make the carrier of thepresent invention includes but are not limited to fascia.

Accordingly, the present invention also provides carrier compositionscomprising a carrier consisting of or consisting essentially of one ormore homogenized connective tissues and optionally, a carrier compoundselected from the group consisting of antioxidants, water miscible polarorganic compounds, water, natural polymers, synthetic polymers,antibiotics, antiviral agents, polysaccharides, extracellular matrix anda combination thereof.

The antioxidant in the carrier composition may be a radioprotectant,such as amino guanidine.

The water miscible polar organic compound may be glycerol.

Biologically active agents used for repairing and/or regeneratingtissues may be added to the carrier composition. Examples of theseagents include but are not limited to bone fragments, blood, bloodproducts, bioactive factors, and/or cells.

The bone fragments may include a plurality of naturally occurring bonefragments, a plurality of synthetic bone fragments, or a combinationthereof. The bone fragments may be demineralized bone fragments,non-demineralized bone fragments, or a combination thereof. Examples ofdemineralized bone fragments include demineralized bone particles and/orfibers. Examples of non-demineralized bone fragments includenon-demineralized cancellous or cortical bone particles and/or fibers.

The addition of the biologically active agents to the carriercomposition of the present invention produces a tissue repaircomposition. In one embodiment, the tissue repair composition of thepresent invention may include a carrier consisting of or consistingessentially of one or more homogenized connective tissues, one or moreantioxidants, one or more water miscible polar organic compounds, one ormore bone fragments, and one or more bioactive factors. In anotherembodiment, the carrier in the tissue repair composition of the presentinvention may consist of or consist essentially of homogenized fascia;the antioxidant in the tissue repair composition may be aminoguanidine;the water miscible polar organic compound may be glycerol; and the bonefragments may be demineralized bone fragments, non-demineralized bonefragments, synthetic bone fragments, or a combination thereof. Thetissue repair composition of the present invention also may serve as acarrier composition for delivering biologically active agents such asbioactive factors or cells to the target site. The target site may be asite of injury or an implant site.

The carrier composition and the tissue repair composition of the presentinvention may be a freeze dried composition.

The present invention provides a method of making a carrier compositionand/or tissue repair composition including (a) preparing a connectivetissue homogenate consisting of or consisting essentially of one or morehomogenized connective tissues; (b) preparing a carrier compositioncomprising mixing the connective tissue homogenate with a carriercompound to produce a carrier composition; and (c) optionally, mixingthe carrier composition with bone fragments and/or biologically activeagents to produce a composition for repairing tissue defects or forhealing wounds. The method of the present invention may further comprisefreeze-drying the carrier composition prior to step (c) or after step(c), but prior to implantation.

The bone fragments used for making the compositions of the presentinvention may be natural demineralized bone fragments, naturalnon-demineralized bone fragments, synthetic bone fragments, or acombination thereof.

The carrier compound used for making the compositions of the presentinvention may comprise a compound selected from the group consisting ofantioxidants, water miscible polar organic compounds, water, naturalpolymers, synthetic polymers, antibiotics, antiviral agents,polysaccharides, and a combination thereof. The antioxidant may be aradioprotectant, such as aminoguanidine, and the water miscible polarorganic compound may be a water replacement agent, such as glycerol. Themethod may further comprise mixing the tissue repair composition with abioactive factor.

The present invention provides a method of promoting tissue repair orregeneration comprising applying a tissue repair composition of thepresent invention to a damaged tissue to promote repair or regenerationof the tissue. The tissue may be an osseous tissue, a cartilage, or asoft tissue. The present invention also include methods of using thetissue repair composition to heal or repair wounds or tissue defects.

The present invention provides a method of inducing bone or cartilageformation comprising applying the tissue repair composition of thepresent invention to an implant site to induce bone or cartilageformation.

The compositions of the present invention, in particular the tissuerepair compositions, may be used for repairing or regenerating varioustissues, such as osseous defect repair, wound repair, cartilage repair,and soft tissue repair.

The present invention provides a coated, prosthetic device comprising animplantable prosthetic device, wherein at least a portion of a surfaceof the implantable prosthetic device is coated with a tissue repaircomposition; wherein the tissue repair composition comprises a pluralityof bone fragments and a carrier composition containing a carrier and oneor more carrier compounds; and wherein the carrier consists essentiallyof one or more homogenized connective tissues.

The present invention also provides a method of coating a prostheticdevice comprising providing an implantable prosthetic device, andapplying a tissue repair composition to at least a portion of a surfaceof the implantable prosthetic device, wherein the tissue repaircomposition comprises a plurality of bone fragments and a carriercomposition containing a carrier and one or more carrier compounds; andwherein the carrier consists essentially of one or more homogenizedconnective tissues.

The tissue repair composition of the present invention may be used forrepairing or healing osseous defects, cartilage defects, and soft tissuedefects. The tissue repair composition of the present invention may alsobe used for wound healing.

DESCRIPTION OF THE INVENTION

The present invention provides a carrier comprising one or morehomogenized connective tissue. The carrier may consist of or consistessentially of one or more homogenized connective tissues. Examples ofconnective tissues that may be used in making the carrier of the presentinvention include but are not limited to fascia, tendons, ligaments,pericardium, cartilage, and mixtures thereof. The carrier of the presentinvention may serve as a vehicle for improved delivery of variousbiologically active agents and/or bone fragments that enhances theireffectiveness at the target site. The target site may be a site ofinjury or an implant site.

The present invention also provides carrier compositions comprising acarrier consisting of or consisting essentially of one or morehomogenized connective tissues and one or more additional carriercompounds. A carrier compound may be selected from the group consistingof antioxidants, water miscible polar organic compounds, water, naturalpolymers, synthetic polymers, antibiotics, antiviral agents,polysaccharides, extracellular matrix and a combination thereof.

Examples of antioxidants include but are not limited to aminoguanidine,ascorbic acid and salts thereof, phytic acid, tocols, isoflavones,vitamin A, β-carotene, selenium, zinc, copper, enzyme superoxidedismutase and its mimetics, or a combination thereof. The ascorbic acidmay be L-ascorbic acid and salts thereof. Examples of tocols includetocopherols and tocotrienols. Examples of isoflavones include soyisoflavones. Selenium may be organic or inorganic selenium. Theantioxidant in the carrier composition may be a radioprotectant, such asamino guanidine.

Examples of water miscible polar organic compounds include but are notlimited to glycerol and ethanol. The water miscible polar organiccompounds may be water replacement agents, such as glycerol.

The extracellular matrix may be extracted from animal tissues.

Biologically active agents used for repairing and/or regeneratingtissues may be added to the carrier composition. Examples of theseagents include but are not limited to bone fragments, blood, bloodproducts, bioactive factors, and/or cells.

The bone fragments may include a plurality of naturally occurring bonefragments, a plurality of synthetic bone fragments, or a combinationthereof. The bone fragments may be demineralized bone fragments,non-demineralized bone fragments, or a combination thereof. Examples ofdemineralized bone fragments include demineralized bone particles and/orfibers. Examples of non-demineralized bone fragments includenon-demineralized cancellous or cortical bone particles and/or fibers.

The blood products included in the carrier composition may include bonemarrow aspirate (BMA), plasma, platelets, plasma rich platelet (PRP),red blood cells, granulocytes, and/or clotting proteins, such as FactorVIII and Factor IX.

Certain embodiments of the present invention are directed tocompositions, such as carrier compositions and tissue repaircompositions, and methods of preparing the same. The compositions of thepresent invention may be in the form of a fluid injectable gel, a fluidinjectable paste, a putty, or a rehydratable freeze-dried paste. Someembodiments of the present invention may be used in clinicalapplications, such as spinal procedures, orthopedic procedures,craniomaxillofacial procedures, and dental procedures. Some embodimentsof the present invention may be used to treat osseous defects, wounds,cartilage defects, and soft tissue defects.

Some embodiments of the present invention are directed to tissue repaircompositions having a carrier consisting or consisting essentially ofone or more homogenized connective tissues, and a plurality of bonefragments. In certain embodiments, the plurality of bone fragmentsincludes demineralized bone fragments, non-demineralized bone fragments,synthetic bone fragments, or a combination thereof. In some embodiments,synthetic bone fragments may contain ceramic material, hydroxyapatite,calcium phosphate, calcium sulfate, calcium carbonate or a combinationthereof. The tissue repair compositions may further include one or morebiologically active agents such as blood products, bioactive factors, orcells. The cells may be mesenchymal stem cells, embryonic stem cells,progenitor cells, differentiated cells, undifferentiated cells, and/orinduced pluripotent stem cells.

In certain aspects of the present invention, the compositions of thepresent invention may include at least one water miscible polar organicsolvent. The water miscible polar organic solvent, for example glyceroland ethanol, may also be a water-replacement agent. In some aspects ofthe present invention, the compositions may include at least oneradioprotectant (i.e., aminoguanidine, among others). In someembodiments, the compositions may include a polymer. In certainembodiments of the present invention, the tissue repair composition maybe freeze-dried.

Some embodiments of the present invention are directed to tissue repaircompositions having a plurality of bone fragments and a carriercomposition. The bone fragments used in the present invention includebut are not limited to demineralized and non-demineralized bonefragments, synthetic bone fragments, or a combination thereof. The bonefragments may be naturally occurring bone fragments or synthetic bonefragments. The bone fragments may also be bone particles or bone fibers.The carrier composition may include a carrier compound and a carrier.The carrier may include one or more homogenized connective tissue. Thecarrier may consist of or consist essentially of one or more homogenizedconnective tissue. The bone fragments may be bone particles or bonefibers, in certain embodiments. In some embodiments, bone fragments maybe derived from allogenic cortical or cancellous bone or xenogeniccortical or cancellous bone. Demineralized bone fragments may, incertain embodiments, have less than about 8 wt % residual calcium orbetween about 0% to 4% residual calcium. The tissue repair compositionmay include between about 5 wt % and about 90 wt % demineralized bonefragments, in some embodiments.

Certain embodiments of the present invention are directed to tissuerepair compositions including a carrier having homogenized fascia, aplurality of bone fragments, one or more radioprotectants, and one ormore water miscible polar organic compounds.

The present invention provides methods of preparing a carriercomposition and a tissue repair composition. The methods of the presentinvention involve preparing a connective tissue homogenate, mixing theconnective tissue homogenate with a carrier compound to obtain a carriercomposition, combining the carrier composition with at least one of aplurality of bone fragments. In some embodiments, the combining stepfurther includes combining the carrier composition and the plurality ofbone fragments with a water miscible polar organic solvent. The watermiscible polar organic solvent may be a water replacement agent. Certainmethods of the present invention involve freeze-drying the carriercomposition before or after the combining step but prior toimplantation. Some aspects of the present invention involvefreeze-drying the carrier and/or the plurality of bone fragments beforethe combining step. Certain methods may further include at least one ofa bone fragmentation step; a connective tissue fragmentation step; aheating step; a connective tissue homogenization step; an optional bonedemineralization step; a selecting of bone fragments, demineralized,non-demineralized, and/or synthetic bone fragments of a particular sizerange step; a freeze-drying step; a packaging step; a sterilizationstep; and a rehydrating step.

Certain methods include combining a plurality of bone fragments and acarrier composition to obtain a tissue repair composition. Optionally,other biologically active agent also be added to such a tissue repaircomposition.

Certain embodiments of the present invention are directed to prostheticdevices including, an implantable prosthetic device, and a coatingdirectly adjacent to at least a portion of a surface of the implantableprosthetic device. The coating includes at least one tissue repaircomposition including (a) a plurality of bone fragments and/or (b) ahomogenized connective tissue.

Some embodiments of the present invention are directed to a method ofcoating a prosthetic device including, providing an implantableprosthetic device, and applying at least one tissue repair compositionto at least a portion of a surface of the implantable prosthetic device.The tissue repair composition is as described herein.

Certain aspects of the present invention are directed to methods ofpromoting tissue repair or regeneration involving applying a tissuerepair composition as described above to a damaged tissue to promoterepair or regeneration of the tissue. The tissue may be an osseoustissue, a cartilage, or a soft tissue. Examples of soft tissues includetendons, ligaments, muscles, synovium, blood vessel, and nerves. Someembodiments of the present invention are directed to methods of inducingbone or cartilage formation involving applying the tissue repaircomposition as described above to an implant site to induce bone orcartilage formation. The tissue repair composition of the presentinvention may also be used to heal or repair wounds or tissue defects.

Some embodiments of the present invention are directed to tissue repairkits that include a carrier having at least one homogenized connectivetissue, and at least one of a plurality of bone fragments. Certainembodiments of the present invention are directed to methods of using atissue repair kit to produce a tissue repair composition. In the presentapplication, the term “connective tissue” refers to mesodermally derivedtissue that may be more or less specialized, and that is, at least inpart, made up of fibers. Most of the connective tissues contemplated inthe present invention are less specialized tissues that are rich inextracellular matrix (i.e., collagen, proteoglycan, among others), andthat surround other more highly ordered tissues and organs. Arelatively, more specialized tissue contemplated in the presentinvention is cartilage. Varieties of connective tissue that may be usedin the present invention include: loose; adipose; dense, regular orirregular; white fibrous; elastic; and cartilage. Connective tissue maybe classified according to concentration of fibers as loose (areolar)and dense, the latter having more abundant fibers than the former.Connective tissues may be obtained from vertebrates. In someembodiments, the tissues may have human, bovine, equine, porcine, ovine,caprine, or piscene origins, among others. Connective tissues may alsobe the product of biotechnological methods (i.e., production of tissueengineered connective tissues using cell culture methods).

Specific examples of connective tissues that may be used in certainembodiments of the present invention include, at least, fascia, tendons,ligaments, pericardium, cartilage, and mixtures thereof. Different typesof fascia that may be used in certain embodiments of the presentinvention include: fascia lata, fascia adherens, fascia brachii, fasciaaxillaris, antebrachial fascia, abdominal fascia, internal fascia,fascia iliaca, fascia profunda, clavipectoral fascia, fascia cribosa,crucial fascia, deltoid fascia, dorsal deep fascia, pelvic fascia,fascia cruris, lumbar fascia, and pectoral fascia, among others.“Crudely fragmented connective tissue” refers to connective tissue thathas been sliced, ground, carved, chipped, chopped, minced, cut,dissected, rent, ripped, sectioned, snipped, diced, shaved, comminuted,or trimmed into fragments having an average diameter greater than about50 microns and less than about 0.5 cm (i.e., having cut dimensions ofapproximately 0.5×0.5 cm), and thickness appropriate to the tissue beingcrudely fragmented. In some embodiments, the crude fragments may not beof uniform size.

A “carrier” is a matrix or substance that may serve as a vehicle toimprove the delivery and maintain the effectiveness of biologicallyactive agents. An example of a carrier that is used in the presentinvention is homogenized connective tissues. The carrier of the presentinvention may comprise one or more homogenized connective tissues. Thecarrier may consist essentially of one or more homogenized connectivetissues.

A “carrier compound” is a compound that is added to a carrier. Examplesof such compounds include but are not limited to antioxidants, watermiscible polar organic compounds, water, natural polymers, syntheticpolymers, antibiotics, antiviral agents, polysaccharides, orextracellular matrix. The antioxidant may be a radioprotectant.

“Homogenized connective tissue” or “connective tissue homogenate”contains connective tissue that has been reduced to particles that areuniformly small and evenly distributed. Homogenized connective tissuemay optionally include at least one of water, aqueous solutions,water-replacement agents, or water miscible polar organic solvents, inaddition to the particles. The homogenized connective tissues used inmethods of the present invention include particles having an averagediameter of less than about 50 microns. In some embodiments, thehomogenized connective tissue may be prepared by shear-induced shreddingof a composition including connective tissue, and optionally, at leastone of water, an aqueous solution, a water-replacement agent, and awater miscible polar organic solvent. A conventional blender may be usedin preparing the homogenized connective tissue, in certain embodiments.

“Osseous tissue” is meant to refer to bone tissue, tissue resemblingbone, and tissue capable of forming bone. The term “bone” or “bonetissue” is intended for the purposes of the invention to refer toautograft bone, allograft bone, and xenograft bone. Such bone includesany bone from any source, including: bone from a living human donor,bone from a human cadaveric donor, and bone from an animal. The bone mayinclude cortical bone and/or cancellous bone and/or cortico-cancellousbone. The term “bone fragment,” as used in the present applicationrefers to ground bone, pulverized bone, bone cubes, bone chips, bonestrips, bone particles, bone rods, and bone fibers. Bone fragments maybe “bone particles” or “bone fibers,” in some embodiments of the presentinvention. “Bone particle” refers to a piece of bone having an averagediameter of between about 125 microns and about 4 mm. “Bone fiber”refers to a filament or thread of bone having an average thickness ofbetween about 0.1 mm and about 1.4 mm and an average width of betweenabout 0.3 mm and about 2.5 mm. Fibers can be of varying lengths. Incertain embodiments, a bone fiber can have an average length of betweenabout 1.0 mm and about 100 mm. In certain embodiments, bone fibercontains lamellae in the shape of threads or filaments having a medianlength to median thickness ratio of about 10:1. “Bone rods” may have anaverage width of between about 0.5 mm and about 5 mm, and an averagelength of between about 1 mm and about 100 mm. “Bone cubes” may have anaverage volume of between about 0.001 mm³ and about 1000 mm³.

Bone fragments used in the present invention may be demineralized ornon-demineralized. “Demineralized bone,” as used in the presentapplication refers to bone having less than about 8 wt % residualcalcium. Demineralization involves treating the surface of a bone tissueto remove a surface layer of its inorganic mineral hydroxyapatitematerial leaving the mechanical properties of the organic phase of thebone constructs substantially unchanged. The level of demineralizationof a bone tissue is defined by the amount (wt %) of residual calciumfound in the demineralized bone. In some embodiments, the demineralizedbone may contain physiologically active levels of growth anddifferentiation factors (i.e., bone morphogenetic proteins (BMPs)).“Non-demineralized bone” as used in the present application refers tobone that has not been treated to remove minerals present such as, forexample, hydroxyapatite.

Bone fragments include “synthetic bone fragments.” The synthetic bonefragments used in the present composition may be composed of one or moreof ceramic, hydroxyapatite, calcium phosphates, calcium sulfates,bioactive glass, and calcium carbonates, among others. The syntheticbone fragments may be in the form of a powder, cubes, chips, strips,particles, rods, and fibers. The synthetic bone fragments may have anaverage diameter of between about 125 microns and about 2000 microns.The synthetic bone fibers may have an average width of between about 0.1mm and about 2 mm, and an average length of between about 0.3 mm andabout 100 mm. The synthetic bone-alternative rods may have an averagewidth of between about 0.5 mm and about 5 mm, and an average length ofbetween about 1 mm and about 100 mm. The synthetic bone alternativecubes may have an average volume of between about 0.001 mm³ and about1000 mm³.

The bone fragments of used in the present invention may include acombination of natural and synthetic bone fragments. The bone fragmentsused in the present invention may also include a combination ofdemineralized and non-demineralized bone fragments.

In the present application, the term “gel” refers to a jelly-like,thick, soft, partly liquid substance. A gel of the present invention maybe extruded through a syringe. The syringe may have a needle of about 13gauge. “Paste,” as used in the present application refers to a soft,moist, substance having a consistency between a liquid and a solid. Apaste of the present invention is less solid than a putty and more solidthat a gel, and in some embodiments may be injectable.

“Putty” refers to a dough-like/clay-like tissue repair composition ofthe present invention. During application the substance may be beaten orkneaded to the consistency of dough, and molded into a shape closelyapproximating that of the implant site.

“Injectable” refers to the ability of certain tissue repair compositionsof the present invention to be introduced at an implant site underpressure (as by introduction using a syringe). An injectable compositionof the present invention may, for example, be introduced betweenelements or into a confined space in vivo (i.e., between pieces of boneor into the interface between a prosthetic device and bone, amongothers).

“Syringe” refers to any device that may be used to inject or withdrawflowable tissue repair compositions of the present invention, includingcertain gels and pastes, among others.

“Flowable” refers to the characteristic of a composition that permits itto be made to fit closely by following the contours of a site. Flowablecompositions may be fluid, malleable, plastic, and/or pliable.

“Allogenic tissue” refers to a tissue from a donor that is implantedinto a recipient of the same species. Allograft tissue is widely used inorthopedic, neuro-, maxillofacial, podiatric, and dental surgery. Thetissue is valuable because it is strong, biointegrates in time with therecipient patient's tissue and may be shaped or reconfigured to fit thespecific surgical defect. Contrasted to most synthetic absorbable ornonabsorbable polymers or metals, allograft tissue is biocompatible andintegrates with the surrounding tissues. Allograft bone occurs in twobasic forms: cancellous and cortical.

“Xenogenic tissue” refers to a tissue from one species that is implantedinto a recipient of another species.

“Cortical bone,” as used in the present application, refers to thecompact bone of the shaft of a bone that surrounds the medullary cavity.Cortical bone is a highly dense structure made up of triple helixstrands of collagen fiber, reinforced with hydroxyapatite. The corticalbone is a compound structure and is the primary load bearing componentof long bones in the human body. The hydroxyapatite component isresponsible for the high compressive strength of the bone while thecollagen fiber component contributes in part to torsional and tensilestrength.

Trabecular bone is of similar composition to cortical bone and is theprimary structural component of “cancellous bone” and refers to adultbone having mineralized regularly ordered parallel collagen fibersorganized differently than in the lamellar bone of the shaft of adultlong bones. Cancellous bone is generally found in the end of long bonessurrounded by cortical bone. Cancellous bone has spicules that form alatticework, with interstices filled with bone marrow. It may also bereferred to as a trabecular bone, or spongy bone.

“Aseptic” as a term can be applied to both products and processes and isgenerally applied to the control or reduction in microbial bioburden.Tissues processed “aseptically” are tissues processed using sterileinstruments, and special environmental surroundings (including forexample “clean room technologies”). Aseptic tissues make reference totissues that are “culture negative,” where culture negative makesreference to the use of representative pieces of tissue that have beenor will be processed for an assessment of the presence ofmicroorganisms. The level of sensitivity of the microbiological testmethod(s), and hence a better definition of “culture negative,” isgenerally predetermined by assessing for interference in the detectionof such microorganisms (sometimes referred to as bacteriostasis andfungistasis, B&F, testing).

“Sterile” makes reference to a definition such as contained in the Codeof Federal Regulations (21 CFR) where the probability of a culturablemicroorganism being present on a processed sample is equal to or lessthan 1 in one million, i.e., a Sterility Assurance Level, or SAL, of1×10⁻⁶.

An “osseous defect” is generally defined by one skilled in the art asbeing an imperfection or void in an osseous tissue, which is ofsufficient physical dimensions as to not heal spontaneously. Hence, theuse of materials generally referred to as “bone void fillers” areutilized clinically to aid or improve healing of the osseous defect.Certain compositions of the present invention can be used as bone voidfillers. Osseous defects may include: fractures, cracks, andosteosarcomas (bone cancer lesions), among others. Bone void fillers maybe used to fill a gap between a prosthetic device and bone; betweenpieces of bone; and between two different prosthetic devices. Forexample, a bone void filler can be used to fill the space between a hipreplacement and a bore in a bone into which the hip replacement has beeninserted.

The tissue repair composition may contain one or more “bioactivefactors”, “bioactive compounds”, “bioactive components” or “bioactivematerials.” Examples of bioactive factors, bioactive compounds,bioactive components, or bioactive materials included in thecompositions of the present invention may be an osteoinductive factor, achondrogenic factor, a cytokine, a mitogenic factor, a chemotacticfactor, a transforming growth factor(TGF), a fibroblast growth factor(FGF), an angiogenic factor, an insulin-like growth factor (IGF), aplatelet-derived growth factor (PDGF), an epidermal growth factor (EGF),a vascular endothelial growth factor (VEGF), a nerve growth factor(NGF), a neurotrophin, a bone morphogenetic protein (BMP), osteogenin,osteopontin, osteocalcin, cementum attachment protein, erythropoietin,thrombopoietin, tumor necrosis factor (TNF), an interferon, a colonystimulating factor (CSF), or an interleukin, among others. The bioactivefactor may be a BMP, PDGF, FGF, VEGF, TGF, insulin, among others.Examples of BMPs include but are not limited to BMP-2, BMP-3, BMP-4,BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13,BMP-14, and BMP-15. The bioactive factor included in the tissue repaircomposition may be a combination of various bioactive factor.

Certain embodiments of the present invention are directed tocompositions having a carrier including at least one homogenizedconnective tissue, and at least one of a plurality of bone fragments.Some embodiments of the present invention are directed to compositionshaving a plurality of bone fragments and a carrier. The carrier mayinclude at least one homogenized connective tissue. In certainembodiments, the carrier may consists of or consist essentially of atleast one connective tissue. In some embodiments, the composition may bea tissue repair composition and may be safely used in repairing damagedosseous tissues (e.g., damaged bone) in an implant patient. The tissuerepair composition may, in some embodiments, be biocompatible,osteoinductive, and/or osteoconductive, such that it may ultimately beremodeled to a mineralized, hard tissue at the application site in vivo.In certain embodiments, the tissue repair compositions may furtherinclude at least one of water, an aqueous solution, a water-replacementagent, a water miscible polar organic solvent, and other componentsdescribed below. The tissue repair composition may include materialsthat improve handling or functional characteristics post-implantation.In some embodiments, bone and connective tissue may be obtained from thesame donor source (i.e., a single human cadaver donor). The formulationof the inventive tissue repair composition may be highly reproducible.In certain embodiments, the tissue repair composition may be aseptic orsterile.

The composition may be in the form of a gel, a paste, a putty, or afreeze-dried substance that can be rehydrated to produce a paste or aputty. In certain embodiments, a freeze-dried substance may berehydrated with a bodily fluid (i.e., blood or bone marrow aspirate,among others). In some embodiments, the gel or paste may be injectable,and the gel or paste may be extrudable through a syringe and/or asyringe having at least a 13 gauge tube/needle coupled thereto. Certaingels and pastes may be used for accurate delivery of the tissue repaircomposition into narrow junctions with minimal surgical damage tosurrounding tissue at the implant site. Some of the tissue repaircompositions of the present invention may be moldable. Tissue repaircompositions of the present invention may be cast into a shaped form, incertain embodiments.

In some embodiments, each of (a) the bone fragments demineralized,non-demineralized, synthetic, or a combination thereof and (b) thehomogenized connective tissue of the inventive composition may includematerials derived from allogenic or xenogenic sources. In certainembodiments, bone and connective tissues obtained from vertebratespecies, for example human, bovine, porcine, equine, ovine, caprine, andpiscene sources may be used to prepare bone fragments and carrier. Theplurality of bone fragments may include more than one type of bonetissue (e.g., cancellous, cortical, or cortico-cancellous bone, anddemineralized or non-demineralized bone), and the connective tissue thatis homogenized to make the carrier may include more than one type ofconnective tissue (i.e., fascia and tendon). The plurality of bonefragments may include bone from a single donor source, or from multipledonor sources, and the homogenized connective tissue may also includetissue from a single donor source, or from multiple donor sources.

For preparation of the tissue repair compositions of the presentinvention, the carrier having the connective tissue homogenate and thebone fragments are combined together. In some embodiments, the tissuerepair composition may include between about 5 wt % and about 90 wt %;between about 20 wt % and about 80 wt %; and between about 30 wt % andabout 50 wt % bone fragments. Certain tissue compositions of the presentinvention that are in the form of a gel or paste may include betweenabout 15 wt % and about 30 wt % bone fragments, while certaincompositions of the present invention that are in the form of a puttymay include between about 25 wt % and about 40 wt % of the bonefragments. In some embodiments, the tissue repair composition may be inthe form of a freeze-dried product that may be rehydrated to produce apaste or a putty, which may include between about 35 wt % and about 70wt % bone fragments.

In certain embodiments, the tissue repair composition may includedemineralized bone fragments having less than about 8 wt %, less thanabout 4 wt % residual calcium, from about 0.5 wt % to about 4 wt %residual calcium, or from about 1 wt % to about 4 wt % residual calcium.The tissue repair composition may include demineralized bone fragmentshaving between about 0 wt % and about 8 wt %, about 0.5 wt % and about 6wt %, about 0.5 wt % and about 4 wt %, or about 2 wt % and 4 wt %residual calcium, in some embodiments.

In certain embodiments, the tissue repair composition may includebetween about 0.25 wt % and about 80 wt %, or about 0.5 wt % and about10 wt % of the connective tissue homogenate. The connective tissuehomogenate may include one or more connective tissues that have beenhomogenized. The amount of homogenized connective tissue used in atissue repair composition may be used to adjust the viscosity andgelation characteristics of the composition.

The plurality of bone fragments may include at least one ofnon-demineralized bone particles, non-demineralized bone fibers,demineralized bone particles, and demineralized bone fibers, in someembodiments. The bone fragments may include materials derived fromallogenic or xenogenic sources. The bone fragments may be derived fromcortical bone or cancellous bone. In certain embodiments, the pluralityof bone fragments includes at least one of non-demineralized allogeniccortical bone particles, non-demineralized xenogenic cortical boneparticles, non-demineralized allogenic cancellous bone particles,non-demineralized xenogenic cancellous bone particles, demineralizedallogenic cortical bone particles, demineralized xenogenic cortical boneparticles, demineralized allogenic cancellous bone particles, anddemineralized xenogenic cancellous bone particles.

Certain tissue repair compositions of the present invention may includebone particles. Bone particles may be prepared from cleaned anddisinfected bone fragments that have been freeze-dried andground/milled/fractured into bone particles. Bone particles may beselected by, for example, using sieving devices (i.e., mesh sieves)commercially available to obtain particles within a desired size range.Such bone particles may have an average diameter of between about 125microns and about 4 mm; between about 125 microns and about 2000microns; between about 710 microns and about 2 mm; between about 125microns and about 500 microns; between about 125 microns and about 850microns; or between about 250 microns and about 710 microns. Certainembodiments of the present invention may include demineralized bonepowder that is commercially available. For example, a suitabledemineralized bone powder that is widely and reliably available isproduced by LifeNet Health, Virginia Beach, Va.

Some tissue repair compositions of the present invention may includebone fibers. Fiber bone may be produced as described in U.S. patentapplication Ser. No. 10/606,208, published as publication number2004/0059364, which is hereby incorporated by reference in its entirety.In certain embodiments, the bone fibers may have an average thickness ofbetween about 0.1 mm and about 0.3 mm and an average width of betweenabout 0.1 mm and about 2 mm; about 0.3 mm and about 1.0 mm; or about 0.3mm and about 2.5 mm. The length of the fibers may vary. In someembodiments, the bone fibers may have an average length of between about0.3 mm and about 100 mm. Bone rods in certain embodiments may have anaverage width of between about 0.5 mm and about 5 mm, and an averagelength of between about 1 mm and about 100 mm. Bone cubes in someembodiments may have an average volume of between about 0.0001 mm³ andabout 1000 mm³.

Any demineralization processes known in the art, may be used to preparedemineralized bone fragments. Examples of such processes are describedin U.S. Pat. Nos. 6,830,763; 6,534,095; 6,305,379; 6,189,537; 5,531,791;and 5,275,954. In some embodiments, the demineralization process beginsby producing bone particles having an average diameter size range ofbetween about 0.1 mm and about 5 mm, about 0.2 mm and about 2 mm, about0.25 and 1 mm, or about 0.25 mm and about 0.75 mm, or by producing bonefibers having an average dimension of 0.1 mm to 0.3 mm thick and anaverage width of about 0.3 mm to about 1 mm. The fragments may then betreated by such processes as are described in U.S. Pat. Nos. 5,556,379;5,797,871; 5,820,581; 5,976,104; 5,977,034; 5,977,432; and 6,024,735,which are hereby incorporated by reference in their entirety. If thebone to be processed into fragments has not been previously cleaned anddisinfected, they may be cleaned and disinfected by the use ofdetergents, hydrogen peroxides, antibiotics, and alcohols to affect aremoval of associated tissues such as bone marrow and cellular elements.Following a cleaning and disinfection, these fragments (i.e., particlesand fibers) may be demineralized by exposure to dilute hydrochloricacid, such as are known in the art, to affect a removal/reduction of themineral component of the bone fragments (i.e., particles and fibers).Such additional processing may, in some instances, inactivate potentialviral contamination (i.e., HIV and hepatitis viruses, among others).

In certain embodiments in which non-demineralized or demineralized bonefragments are to be used later, they may be conveniently stored byfreeze-drying, which may maintain the activity of their bioactivecomponents (i.e., BMPs, among others). If the bone fragments are to beused later, in some embodiments, the acidic demineralization solutionmay be removed from the bone using aqueous or polar (miscible withwater) organic solutions, for example deionized/distilled endotoxin-freewater, saline solutions, acetone, alcohol(s), and dimethylsulfoxide, inorder to minimize elevated levels of salts in the freeze-dried bone.

Some tissue repair compositions of the present invention may includesynthetic bone fragments. Such synthetic bone fragments may includefragments having an average diameter of between about 125 microns andabout 2000 microns, or between about 250 microns and about 710 microns.The synthetic bone fragments may include synthetic bone fibers having anaverage thickness of between about 0.1 mm and about 0.3 mm and anaverage width of between about 0.1 mm and about 2 mm, or about 0.3 mmand about 2.5 mm. The length of the fibers may vary. In someembodiments, the bone-alternative fibers may have an average length ofbetween about 0.3 mm and about 100 mm. Synthetic bone fragments mayinclude bone rods in certain embodiments having an average width ofbetween about 0.5 mm and about 5 mm, and an average length of betweenabout 1 mm and about 100 mm. Synthetic bone fragments may include bonecubes in some embodiments having an average volume of between about0.0001 mm³ and about 1000 mm³.

Compositions of the present invention may contain a carrier having ahomogenized connective tissue. In certain embodiments, the carrier inthe composition may consist of or consist essentially of homogenizedconnective tissue. In other embodiments, the composition may contain acarrier along with one or more other components. The components may be acarrier compound and/or a biologically active agent, such as bonefragments. In some embodiments, the carrier may include a biocompatibleliquefied form of connective tissue (i.e., liquefied human connectivetissue) that when combined with bone fragments, has suitable viscosityso as to be injectable through large gauge applicators, while largelyremaining at the implant site. The carrier may promote cellularinfiltration and retain the bone fragments at the site of application,without being cytotoxic. The carrier may promote such cellularinfiltration by providing a molecular matrix for cell migration. In someembodiments, the carrier may be freeze-dried prior to combining withother components. In certain embodiments, the carrier includeshomogenized fascia.

In some embodiments, the homogenized connective tissue may be preparedfrom allogenic or xenogenic tissue. Such connective tissue may beobtained from a human donor or an animal (i.e., bovine donor, porcinedonor, etc.). Connective tissue may be obtained relatively economically.Varieties of connective tissue that may be used in certain embodimentsof the present invention include: areolar or loose; adipose; dense,regular or irregular; white fibrous; elastic; and cartilage. Specificexamples of connective tissues that may be used in certain embodimentsof the present invention include, at least: fascia, tendons, ligaments,pericardium, cartilage, and combinations thereof. Different types offascia that may be used in some embodiments of the present inventioninclude: fascia lata, fascia adherens, fascia brachii, fascia axillaris,antebrachial fascia, abdominal fascia, internal fascia, fascia iliaca,fascia profunda, clavipectoral fascia, fascia cribosa, crucial fascia,deltoid fascia, dorsal deep fascia, pelvic fascia, fascia cruris, lumbarfascia, and pectoral fascia, among others. For practical reasons ofavailability during procurement and amount of fascia available, fascialata from the anterior portion of the upper leg may be used in certainembodiments.

Homogenized connective tissue may be prepared by methods involving,cleaning and disinfecting connective tissue, and removing extraneoustissues associated with the connective tissue. Connective tissues may becut into small pieces to produce crudely fragmented connective tissue,and optionally triturated and washed with distilled/deionizedendotoxin-free water and/or an aqueous solution (i.e., isotonic saline,among others). In processing, multiple “washes” may be affected usingvolumes of aqueous solution that are 10 times the approximated volume ofthe tissue being processed, in some embodiments. It would be obvious toone skilled in the art that the use of three such processing steps wouldaffect an approximate 1:1000 dilution of associated solubilizableelements rendering the tissue essentially free from such solubilizableelements.

Connective tissue may be treated and homogenized at temperaturessufficient to produce a flowable homogenized connective tissue, incertain embodiments. The homogenized connective tissue may includeconnective tissue that has been reduced to particles that are uniformlysmall and evenly distributed. Homogenized connective tissue and/or thecarrier may optionally include at least one of water, aqueous solutions(i.e., isotonic saline), water-replacement agents, and water misciblepolar organic solvents in addition to the connective tissue particles.In some aspects, the homogenized connective tissue may include gelatin.The homogenized connective tissues used in methods of the presentinvention may include particles having an average diameter of less thanabout 50 microns, less than about 20 microns, or less than about 50microns and greater than about 5 microns. In some embodiments, thehomogenized connective tissue and optionally, at least one of awater-replacement agent, a water miscible polar organic solvent, water,and an aqueous solution, may be prepared by shear-induced shredding ofconnective tissue. A conventional blender may be used in preparing thehomogenized connective tissue, in certain embodiments.

In some embodiments of the present invention, connective tissuehomogenate and/or the carrier will retain large and small molecularweight macromolecules, including hyaluronate which is known to play arole in cell migration (Toole, B. P. and Trelstad, R. L., 1971, Develop.Biol. 26:28-35; Docherty, R., et al., 1989, J. Cell. Sci. 92:263-270)and has been implicated in facilitating fibril formation which promotesgelation (Tsunenaga, M., et al., 1992, Connect. Tiss. Res. 28:113-123).The connective tissue homogenate and/or carrier may have excellenthistocompatibility and elicit minimal antibody formation orimmunological rejection, in certain embodiments. Keeping this in mind,the homogenized connective tissue may be made acellular, using methodsknown in the art, prior to homogenization, and methods of making suchtissues acellular are described in U.S. Pat. Nos. 6,734,018 and6,743,574; which are hereby incorporated by reference in their entirety.

In certain embodiments, the homogenized connective tissue may beacellular or cellular. An acellularization process used to preparehomogenized connective tissue of the present invention may be performedwithout damage to matrix and/or tissue structure, in some embodiments.Mechanical strength of a connective tissue may reside in the matrixstructure of the tissue. The matrix structure may include collagens,hyaluronins, elastins, mucopolysaccharides and proteoglycans, amongother components. An example of an acellularization method for use withsoft tissues is described in U.S. Pat. Nos. 6,734,018 and 6,743,574,which are hereby incorporated by reference in their entirety. Connectivetissue that is acellularized may have a thickness that does not exceedabout 8 mm, about 6 mm, about 4 mm, about 2 mm, about 1 mm, or about 0.2mm, in certain embodiments. Acellularization processing may be alteredto accommodate the thicker tissues.

The compositions of the present invention may contain additionalelements such as antioxidants, polymers, bodily fluids (i.e., blood orbone marrow aspirate, among others), water, aqueous solutions,water-replacement agents, water miscible polar organic solvent,surfactants, bioactive factors, antibiotics (i.e., penicillin),antiviral agents (i.e., Triton X-100, Nonidet P40, N-lauroylsarcosinate, Brij-35, and peroxide generating agents), antitumor agents,analgesics, immunosuppressive agents (i.e., bovine intestinal alkalinephosphatase), permeation enhancers (i.e., fatty acid esters, such as thelaurate, myristate and stearate monoesters of polyethylene glycol),nucleic acids, mesenchymal elements, gelation enhancing compounds (i.e.,hyaluronic acid, chondroitin sulfate, dermatin sulfate,carboxymethylcellulose, methylcellulose, polyethylene glycol, orglycosamino glycans), or autogenously derived osteoprogenitor cellsand/or osteoblast cells, and other biologically active agents. Somecompounds may fall into more than one category of additional elements.For example, glycerol may be both a water-replacement agent and a watermiscible polar organic solvent. As another example, Triton X-100 may beboth a surfactant and an antiviral agent.

Antioxidants such as radioprotectants may be included in the compoundsof the present invention. Radioprotectants may provide protectionagainst certain undesirable effects of ionizing radiation. In someembodiments, a composition of the present invention, such as a tissuerepair composition, may be subjected to irradiation, and the tissuerepair composition may include at least one radioprotectant, for exampleaminoguanidine, that reduces undesirable effects (i.e., poor handlingcharacteristics among others) that may result from such irradiation. Insome embodiments, a radioprotectant may be biocompatible, and that theradioprotectant itself may not introduce undesirable properties into atissue repair composition. In certain embodiments of the presentinvention, the tissue repair composition may include a radioprotectant,and may be osteoinductive and biocompatible. In some embodiments, thetissue repair composition may include between 10 mM and about 500 mMradioprotectant(s).

Exemplary radioprotectants that may be used in some embodiments of thepresent invention include aminoguanidine, ascorbic acid and saltsthereof, and phytic acid and salts thereof. In certain embodiments, theradioprotectant may be aminoguanidine at a concentration between about10 mM and about 1M, or between about 10 mM and 100 mM in the tissuerepair composition. In some embodiments of the present invention, theradioprotectant may be L-ascorbic acid salt at a concentration ofbetween about 50 μM and about 1M, between about 150 mM and about 500 mM,or between about 250 mM and about 500 mM in the tissue repaircomposition. In certain embodiments, the radioprotectant may beL-ascorbic acid 2-phosphate sesquimagnesium salt at a concentration ofbetween about 50 μM and about 1M, between about 50 μM and about 500 mM,or between about 50 mM and about 250 mM. In some embodiments, theradioprotectant may be phytic acid at a concentration of between about100 μM and about 100 mM in the tissue repair composition. Aradioprotecant may be added to the composition of the present invention,such as a tissue repair composition, or a component of a composition atany point during processing before the composition or component isirradiated, in certain embodiments.

Polymers that may be used in certain tissue repair compositions of thepresent invention include natural polymers and synthetic polymers. Incertain embodiments of present invention, a polymer in a tissue repaircomposition may be a polysaccharide, such as alginate, propylene glycolalginate, native or crosslinked chitosan, starch, agarose, chitin,cellulose and derivatives thereof (i.e., cellulose acetate,carboxymethyl cellulose, and methylcellulose, among others), xanthangum, dextran, carrageenan, hyaluronic acid, chondroitin sulfate, locustbean gum, gum tragacanth, gum arabic, curdlan, pullulan, scleroglucan,or lower methoxyl pectin, among others. In some embodiments, a tissuerepair composition of the present invention may include at least one ofnative or modified extracellular matrix, such as collagen, gelatin,hyaluronic acid, fibrin, MATRIGEL™ (BD Biosciences, San Jose, Calif.),human extracellular matrix (i.e., a chromatographically partiallypurified matrix extract derived from human placenta is available from BDBiosciences, San Jose, Calif.), proteoglycans, laminin, fibronectin, orelastin. In some embodiments, a tissue repair composition of the presentinvention may include heparin, polyethylene glycol, biotin, avidin,polymethylmethacrylate, polyurethane, acryloilmorpholine, N,N-dimethylacrylamide, N-vinyl pyrrolidone, glycerol, sucrose octasulfate,hydroxyapatite, tetrahydrofurfuryl methacrylate, and polylactic acid.Some tissue repair compositions of the present invention may includecross-linkage or functionalization of hyaluronan, collagen, or alginate.

Water-replacement agents that may be used in certain embodiments of thepresent invention include glycerol (glycerin USP), adonitol, sorbitol,ribitol, galactitol, D-galactose, 1,3-dihydroxypropanol, ethyleneglycol, triethylene glycol, propylene glycol, glucose, sucrose,mannitol, xylitol, meso-erythritol, adipic acid, proline,hydroxyproline, polyethylene glycol, alcohol, and lipids. Certain tissuerepair compositions of the present invention include glycerol.

Examples of bioactive factors include: bone morphogenic proteins,transforming growth factor beta, fibroblast growth factor, insulin,vascular endothelial growth factor, and platelet derived growth factor,among others. In this respect, the invention includes other equivalentoptional components readily known to those in the art.

Tissue repair compositions of the present invention may include acalcium phosphate and/or calcium sulfate mineral component to produce anosteoinductive/osteoconductive composition which will harden prior to orpost implantation. Tissue repair compositions of the present inventionmay also include particulate hydroxyapatite, calcium phosphate,magnesium phosphate, calcium carbonate, as extenders of the compositionsand as sources of mineral in subsequent induced new bone formation.

As described above, certain tissue repair compositions of the presentinvention may include a plurality of bone fragments and a carrier. Thecarrier may consist of or consist essentially of at least onehomogenized connective tissue, and, optionally, one or more additionalcomponents. The carrier may consist essentially of one or morehomogenized connective tissue. In some embodiments of the presentinvention, no water is added to the carrier/homogenized connectivetissue. In certain carriers of the present invention, the weight ratioof the additional component(s) to the wet weight of homogenizedconnective tissue(s) may be from about 0.1:1 to about 12:1, or fromabout 0.5:1 to about 6:1. In some embodiments, the at least oneadditional component may be water, an aqueous solution, alginate,propylene glycol alginate, native or crosslinked chitosan, starch,cellulose and derivatives thereof, xanthan gum, dextran, carrageenan,hyaluronic acid, chondroitin sulfate, locust bean gum, gum tragacanth,gum arabic, curdlan, pullulan, scleroglucan, lower methoxyl pectin,native or modified collagen, gelatin, agarose, modified hyaluronic acid,fibrin, chitin, biotin, avidin, Matrigel™, human extracellular matrix,proteoglycans, laminin, fibronectin, elastin, heparin, polyethyleneglycol, polymethylmethacrylate, polyurethane, acryloilmorpholine,N,N-dimethyl acrylamide, N-vinyl pyrrolidone, glycerol, sucroseoctasulfate, hydroxyapatite, tetrahydrofurfuryl methacrylate, orpolylactic acid. In certain embodiments of the present invention, theadditional component in a carrier may include a water-replacement agentor a water miscible polar organic solvent, as described above. In someembodiments, the additional component may be about 0% to about 100%water by weight, or about 0.1% to about 90% water by weight. Thus, ifthe additional component is water, it is 100% water by weight, and ifthe additional component is a 10 wt % glycerol solution, it is 90% waterby weight.

Certain embodiments of the present invention are directed to tissuerepair compositions including a carrier including homogenized fascia, aplurality of bone fragments, at least one radioprotectant, and at leastone water-replacement agent or at least one water miscible polar organicsolvent. In some embodiments, the carrier consists of homogenizedfascia. In certain embodiments the bone fragments may be demineralizedbone fragments, non-demineralized bone fragments, synthetic bonefragments, or a combination thereof. The bone fragments may befreeze-dried bone fragments in some embodiments of the presentinvention. In certain aspects of the present invention, thewater-replacement agent may be glycerol. The radioprotectant may beaminoguanidine, and the tissue repair composition may include betweenabout 10 mM and about 100 mM aminoguanidine, in some embodiments.

Some embodiments of the present invention are directed to tissue repairkits including: a carrier having at least one homogenized connectivetissue, and at least one of a plurality of bone fragments. In certainaspects of the present invention, the carrier consists essentially of atleast one homogenized connective tissue and a water-replacement agent.The carrier may be a freeze-dried carrier in some embodiments of thepresent invention. In certain embodiments, the bone fragments in the kitmay be freeze-dried. In some tissue repair kits of the presentinvention, the bone fragments include a water-replacement agent. In someembodiments, the water-replacement agent may be glycerol.

In some embodiments, the tissue repair composition or components of atissue repair composition, and optionally means for applying a tissuerepair composition (i.e., syringe or spatula) to an implant site may beprovided in a unitary kit. In other embodiments, the bone fragments, theconnective tissue homogenate, and/or the carrier may be prepared understerile conditions and stored separately, or mixed and stored together,for later use. To facilitate clinical usage of described tissue repaircompositions, the bone fragments and/or the bone-alternative fragments,and the carrier/connective tissue homogenate may be packaged separatelyin different forms and reconstituted and combined at the time of usage,in some embodiments. In other embodiments, the components may becombined to produce a tissue repair composition, which is then packaged,in a premixed format.

Certain embodiments of the present invention are directed to methods ofusing the tissue repair compositions, described above. Some methods ofthe present invention include combining the carrier and the plurality ofbone fragments in a tissue repair kit to produce a tissue repaircomposition. In certain embodiments, the methods further include addingat least one of water, an aqueous solution, and a bodily fluid to atleast one component of the kit (e.g., the carrier and/or the pluralityof bone fragments), or a tissue repair composition prepared using thekit.

The premixed format provides the advantage of requiring minimalpreparation by the individual clinician at the time of usage. In someembodiments, the tissue repair composition may be stored in anapplication means, such as a syringe, which will be used to apply thecomposition to an osseous defect site. The tissue repair compositionmay, for example, be stored in a 1 to 10 cc syringe that is capable ofbeing coupled to a large gauge delivery tube/needle of appropriatelength and inside diameter. In this regard, a delivery tube with aninside diameter of not less than 13 gauge is appropriate for theinjection delivery into an implant site.

For on-site preparation, the carrier/homogenized connective tissue andbone may be provided in freeze-dried aliquots that are rehydrated justprior to being combined for use in clinical applications, in someembodiments. In some embodiments, the aliquots may be rehydrated with abodily fluid (i.e., blood or bone marrow aspirate, among others).On-site preparation has the advantage of increasing the ability to varythe concentrations and quantities of the connective tissue homogenateand/or bone fragments used in preparation of the inventive tissue repaircomposition. Furthermore, on-site preparation permits the addition ofoptional components at the discretion of the clinician.

Certain embodiments of the present invention are directed to methods forthe preparation of the tissue repair compositions described above. Suchmethods include combining a plurality of bone fragments and a carrierhaving a homogenized connective tissue. Certain inventive methodsinclude combining bone fragments with a carrier such that the tissuerepair composition produced between about 5 wt % and about 90 wt %;between about 20 wt % and about 80 wt %; and between about 30 wt % andabout 50 wt % bone fragments. In some embodiments, the bone fragmentsand the carrier may be combined with at least one of the component, asdescribed above. The methods may include packaging the inventive tissuerepair compositions, in certain embodiments. In some embodiments,inventive methods of the present invention include providing at leastone of bone tissue or bone tissue fragments and at least one connectivetissue, and preparing bone fragments and homogenized connective tissuefrom the at least one bone tissue and the at least one connectivetissue, as described above. Bone fragments may include at least one ofbone particles and bone fibers from bone tissue. Bone fragments mayinclude demineralized bone fragments, non-demineralized bone fragment,synthetic bone fragments, or a combination thereof. Some methods ofpreparing the tissue repair compositions of the present invention mayinclude the production of particles or fibers from bone tissue, asdiscussed above. Bone fragments may be demineralized, as describedabove, in certain embodiments. The bone fragments may be demineralizedto have less than about 8 wt % residual calcium, less than about 4 wt %residual calcium, from about 0.5 wt % to about 4 wt % residual calcium,or from about 1 wt % to about 4 wt % residual calcium. The bonefragments may be demineralized to have between about 0 wt % and about 8wt %, about 0.5 wt % and about 6 wt %, about 0.5 wt % and about 4 wt %,or about 2 wt % and about 4 wt % residual calcium, in some methods ofthe present invention. Certain methods of the present invention mayinclude freeze-drying bone fragments. In some embodiments, the bonefragments may be freeze-dried to a point such that the freeze-driedfragments have an average residual moisture of less than about 10 wt %,or less than about 5 wt %. In some embodiments, freeze-dried bonefragments may be rehydrated before use in preparing the tissue repaircompositions of the present invention.

Certain methods for producing the compositions of the present inventionmay include preparing a connective tissue homogenate/carrier. Prior tohomogenization, connective tissues (i.e., fascia, tendons, ligaments,pericardium, and cartilage, among others) may be crudely fragmented.Connective tissue (e.g., fresh or freeze-dried) may be sliced, ground,carved, chipped, chopped, minced, cut, dissected, rent, ripped,sectioned, snipped, diced, shaved, comminuted, or trimmed into crudefragments. In some embodiments, the crude fragments may have an averagediameter greater than about 50 microns. The crude fragments may be ofvarying sizes, in some embodiments. Essentially intact connective tissueor crude fragments of connective tissue (e.g., fresh or freeze-dried)may be homogenized at least one time to prepare the homogenate. Thehomogenization step(s) of certain inventive methods may involveshear-induced shredding of connective tissue. Connective tissue may behomogenized to have tissue fragments having an average diameter of lessthan about 50 microns, less than about 20 microns, or less than about 50microns and more than about 5 microns. Water, at least one aqueoussolution (e.g., isotonic saline), a water-replacement agent, or othercomponents may be combined with a connective tissue beforehomogenization.

Certain methods include at least one of (a) heating a connective tissuebefore it is homogenized, (b) heating a connective tissue while it isbeing homogenized, and (c) heating a connective tissue homogenate. Insome embodiments the heating is done to a temperature of between aboutambient temperature and about 100° C., or between about 37° C. and about100° C. The heating may be carried out for between about 4 minutes andabout 30 minutes. The heating may be accomplished using sonication,microwave irradiation, or conventional heat transfer from a heatingcomponent, among other methods known in the art.

In certain methods, the tissue repair composition may be cast in a mold.In some embodiments, a method may further include freeze-drying a castcomposition or cross-linking a cast composition utilizing chemicalreagents known in the art.

Methods of the current invention may include sterilization of tissuerepair compositions, components of tissue repair compositions, and/orsterilization of packaged tissue repair compositions/components.Sterilization may be performed using methods known in the art. Thesterilization may involve the use of ionizing radiation, in someembodiments. Methods of the current invention may include irradiation oftissue repair compositions, components of tissue repair compositions,and/or irradiation of packaged tissue repair compositions/components.Irradiation may be performed using methods known in the art. Theirradiation may involve the use of ionizing radiation, in someembodiments. In certain embodiments, the absorbed dose of ionizingradiation may be between about 8.0 kGy and about 50 kGy, between about8.0 kGy and about 25 kGy, and between about 8.0 kGy and about 18 kGy. Insome embodiments, the sterilizing and/or irradiation step includesplacing the packaged composition on dry ice and irradiating the packagedcomposition. In certain embodiments, sterilization and/or irradiationmay be performed at a temperature of between about −20° C. and −50° C.In some embodiments of the present invention, the composition orcomponent of a tissue repair composition that is irradiated after it hasbeen freeze dried.

Certain methods of the present invention involve (a) providing at leastone connective tissue and at least one bone tissue from at least onecadaver, (b) freeze-drying the connective tissue, (c) crudelyfragmenting the connective tissue, (d) adding at least one of water, anaqueous solution (i.e., isotonic saline), a water-replacement agent, ora water miscible polar organic solvent to the crude fragments to producea mixture, which may optionally be heated, (e) homogenizing the mixtureto produce a connective tissue homogenate, (f) fragmenting the bonetissue to produce fragments, (g) optionally demineralizing the bonefragments, (h) freeze-drying the bone fragments, (i) selecting bonefragments having sizes within a particular range, (j) combining theselected bone fragments of the particular range with the connectivetissue homogenate. Certain methods may include at least one of (a)heating a connective tissue before it is homogenized, (b) heating aconnective tissue while it is being homogenized, (c) heating aconnective tissue homogenate, and (d) heating the tissue repaircomposition. In some embodiments, heating is sufficient to reach atemperature of about 100° C. A microwave oven may be used in the heatingstep, in certain embodiments. Connective tissue homogenate may be heatedand homogenized a second time before being combined with the bonefragments in certain methods of the present invention. In someembodiments, the selecting of bone fragments having sizes with a givenrange may involve the use of mesh sieves. In some embodiments, thetissue repair composition may be packaged, and the packaged compositionmay optionally be sterilized.

Certain embodiments of the present invention are directed to methods forproducing a tissue repair composition including: (a) producing aconnective tissue homogenate from one or more connective tissues; (b)mixing the connective tissue homogenate with at least one of water, anaqueous solution, a water-replacement agent, a bodily fluid, or a watermiscible polar organic solvent to produce a carrier; (c) mixing at leastone or more bone fragments with the carrier to produce a tissue repaircomposition; and (d) freezing or freeze-drying the tissue repaircomposition.

Some embodiments of the present invention are directed to methods forproducing a tissue repair composition including: (a) producing aconnective tissue homogenate from one or more connective tissues; (b)mixing the connective tissue homogenate with at least one of water, anaqueous solution, a water-replacement agent, a bodily fluid, or a watermiscible polar organic solvent to produce a carrier; (d) freezing orfreeze-drying the carrier; and (e) mixing at least one or more of thebone fragments with the freeze-dried carrier to produce a tissue repaircomposition. In certain embodiments, the freeze-dried carrier may bemixed with the bone fragments and packaged. In some embodiments, thefreeze-dried carrier may be packaged separately, or as a kit with thebone fragments, which may be mixed right before implantation or surgery.

The steps set forth above for preparing inventive tissue repaircompositions may be combined and the sequence of steps may be changed.In some embodiments, water, an aqueous solution, a water-replacementagent, or a water miscible polar organic solvent may be added to aconnective tissue as it is homogenized to prepare a homogenate ofconnective tissue.

In certain embodiments, inventive tissue repair compositions of thepresent application may be applied to a prosthetic device utilized inneurological or orthopedic applications, to facilitate osteoconduction,and/or osteoinduction of native bone around the implant in order tobuild a stronger and more compatible association between the implant andthe native bone. Implantable bone prostheses may include a substrateformed of a biocompatible metal, ceramic, mineral component, orcomposite; and at least a partial coating of tissue repair composition.

Certain embodiments of the present invention are directed to coatedprosthetic devices including, an implantable prosthetic device, and acoating directly adjacent to at least a portion of a surface of theimplantable prosthetic device. The coating includes at least one tissuerepair composition including (a) a plurality of bone fragments and (b) ahomogenized connective tissue.

Some embodiments of the present invention are directed to a method ofcoating a prosthetic device including, providing an implantableprosthetic device, and applying at least one tissue repair compositionto at least a portion of a surface of the implantable prosthetic device.The tissue repair composition is as described above.

Certain embodiments of the present invention are directed to methods ofpromoting tissue repair or regeneration including applying a tissuerepair composition as described above to a damaged tissue to promoterepair or regeneration of the tissue. The tissue may be an osseoustissue, a cartilage, or a soft tissue. Soft tissues may include but arenot limited to tendons, ligaments, muscles, synovium, blood vessels, andnerves. Some embodiments of the present invention are directed tomethods of inducing bone or cartilage formation including applying atissue repair composition as described above to an implant site toinduce bone or cartilage formation. The present invention also providesmethods of using the tissue repair composition to heal or repair woundsor tissue defects.

The bioactive factor included in the compositions of the presentinvention may be an osteoinductive factor, a chondrogenic factor, acytokine, a mitogenic factor, a chemotactic factor, a transforminggrowth factor(TGF), a fibroblast growth factor (FGF), an angiogenicfactor, an insulin-like growth factor (IGF), a platelet-derived growthfactor (PDGF), an epidermal growth factor (EGF), a vascular endothelialgrowth factor (VEGF), a nerve growth factor (NGF), a neurotrophin, abone morphogenetic protein (BMP), osteogenin, osteopontin, osteocalcin,cementum attachment protein, erythropoietin, thrombopoietin, tumornecrosis factor (TNF), an interferon, a colony stimulating factor (CSF),or an interleukin, among others. The bioactive factor may be a BMP,PDGF, FGF, VEGF, TGF, insulin, among others. Examples of BMPs includebut are not limited to BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8,BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, and BMP-15. The bioactivefactors included in the tissue repair compositions of the presentinvention may be a combination of one or more bioactive factors.

In one embodiment, the bioactive factor may be a growth factor selectedfrom the group consisting of transforming growth factor beta, afibroblast growth factor, a vascular endothelial growth factor, or aplatelet derived growth factor. In another embodiment, the bioactivefactor is a bone morphogenic protein or insulin.

The composition of the present invention may comprise bone fragments atbetween about 5 wt % and about 90 wt %, about 20 wt % and about 80 wt %,or about 30 wt % and about 50 wt %. The bone fragments may have anaverage diameter of between about 125 microns and about 4 mm, about 125microns and 850 microns, about 125 microns and about 710 microns, about125 microns and about 500 microns, about 250 microns and about 710microns.

The demineralized bone fragments may have less than about 8 wt %residual calcium, less than about 4 wt % residual calcium, from about0.5 wt % to about 4 wt % residual calcium, or from about 1 wt % to about4 wt % residual calcium. The demineralized bone fragments may havebetween about 0 wt % to about 8 wt %, about 0.5 wt % and about 6 wt %,about 0.5 wt % and about 4 wt %, or about 2 wt % and about 4 wt %residual calcium.

The compositions of the present invention may include homogenizedconnective tissue in the amount of between about 0.25 wt % and about 80wt %, about 0.5 wt % and about 5 wt %, or about 0.5 wt % and about 4 wt%. The homogenized connective tissue may be homogenized fascia,homogenized tendon, homogenized ligament, homogenized pericardium,homogenized cartilage, or a mixture thereof. The homogenized connectivetissue may be acellular.

In one embodiment, the composition of the present invention may comprisebone fragments from a single donor source or multiple donor sources. Inanother embodiment, the composition of the present invention maycomprise homogenized connective tissue from a single donor source ormultiple donor sources. The composition of the present invention mayalso comprise an osteoconductive component.

The compositions of the present invention may also include a calciumphosphate mineral and a calcium sulfate mineral. The mineral may hardenprior to or post implantation.

The composition of the present invention may also include hydroxyapatiteparticulates, calcium phosphate particulates, magnesium phosphateparticulates, and calcium carbonate particulates.

The compositions of the present invention may further include corticalbone fragments, cancellous bone fragments and ground/milled corticalbone fragments.

The present invention provides a coated, prosthetic device comprising animplantable prosthetic device, wherein at least a portion of a surfaceof the implantable prosthetic device is coated with a composition of thepresent invention, such as a tissue repair composition. The tissuerepair composition may comprise a plurality of bone fragments, or acombination thereof, and a carrier composition containing a carrier andone or more carrier compounds; and wherein the carrier consists of orconsists essentially of one or more homogenized connective tissues.

The present invention also provides a method of coating a prostheticdevice comprising applying a composition of the present invention, suchas a tissue repair composition, to at least a portion of a surface ofthe implantable prosthetic device, wherein the tissue repair compositioncomprises a plurality of bone fragments, and a carrier compositioncontaining a carrier and one or more carrier compounds; and wherein thecarrier consists essentially of one or more homogenized connectivetissues.

Moreover, the present invention provides implanting a coated, prostheticdevice at an osseous implant site wherein the method includes preparingan osseous implant site, and implanting the coated, prosthetic device ofthe present invention at the implant site.

In one embodiment, the method of coating the prosthetic device furthercomprises freeze-drying the tissue repair composition on the surface ofthe implantable prosthetic device. Accordingly, the present inventionmay also include a coated prosthetic device, wherein the tissue repaircomposition is freeze-dried onto the surface of the implantableprosthetic device. In another embodiment, the method further comprisesfreeze-drying the tissue repair composition prior to applying it to thesurface of the prosthetic device.

The method may comprise applying a chemical reagent to the surface ofthe prosthetic device. The method may also include shaping the tissuerepair composition using a mold after applying the tissue repaircomposition to the surface of the prosthetic device. Moreover, themethod may include freeze-drying the tissue repair composition aftershaping the tissue repair composition.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the claimed invention. Thefollowing working examples therefore, specifically point out embodimentsof the present invention, and are not to be construed as limiting in anyway the remainder of the disclosure. All articles, publications, patentsand documents referred to throughout this application are herebyincorporated by reference in their entirety.

Further details of the process of the invention are presented in theexamples that follow:

EXAMPLES Example 1 Preparation of Tissue Repair Compositions ContainingFreeze-Dried Fascia

Fascia lata and bone from a human cadaver were procured and returned tothe processing facility under sterile conditions. Donor histories,personal and medical, were obtained following accepted standards of theAmerican Association of Tissue Banks Microbiological tests wereperformed following FDA guidelines for testing sterility of products.

The bone and fascia were cleaned of unwanted tissues and freeze-dried.The freeze-dried fascia was cut into small (about ½ cm by ½ cm) pieces(e.g., crude fragments). Isotonic saline in a volume (1 cm³ of isotonicsaline corresponds to about 1 g) approximating 20 times the weight oftissue, was added to a container containing the cut fascia. Theingredients were heated to a temperature of 100° C. using a microwaveoven, and maintained at this temperature for 4 minutes. Water was addedto the heated composition to replace the liquid lost to evaporation. Theheated composition was transferred into a conventional blender andmechanically homogenized (e.g., blended) for 5 minutes. The homogenizedconnective tissue was re-heated for an additional 4 minutes in themicrowave oven, and mechanical homogenization was repeated for anadditional 5 minutes (e.g., until the mixture was liquefied andhomogeneous).

Ground demineralized bone powder was prepared by impact fragmentation ofbone, followed by freeze-drying. The freeze-dried particles were sizedusing mesh sieves. Ground demineralized bone particles having a size inthe range of about 250 to 710 microns and demineralized to an averageweight percent residual calcium of 2±1% were used.

To prepare a first tissue repair composition, the sized, freeze-drieddemineralized bone powder was added to the homogenized fascia until thefinal concentration of the bone was about 30% by weight. In a secondtissue repair composition, the demineralized bone powder was added tothe homogenized fascia until the final concentration of the bone wasabout 65% by weight. Samples of the tissue repair composition weresealed in sterilized glass vials in 2 g aliquots.

Example 2 Preparation of a Tissue Repair Composition ContainingFreeze-Dried Tendon

Tendon and bone from a human cadaver were procured and returned to theprocessing facility under sterile conditions. Donor histories, personaland medical, were obtained following accepted standards of the AmericanAssociation of Tissue Banks Microbiological tests were performedfollowing FDA guidelines for testing sterility of products.

The bone and tendons were cleaned of unwanted tissues and freeze-dried.The freeze-dried tendon was cut into small (about ½ cm by ½ cm) pieces(e.g., crude fragments). Isotonic saline in a volume (1 cm³ of isotonicsaline corresponds to about 1 g) approximating 20 times the weight oftissue, was added to a container containing the cut fascia. Theingredients were heated to a temperature of 100° C. using a microwaveoven, and maintained at this temperature for 4 minutes. Water was addedto the heated composition to replace the liquid lost to evaporation. Theheated composition was transferred into a conventional blender andmechanically homogenized (e.g., blended) for 5 minutes. The homogenizedconnective tissue was re-heated for an additional 4 minutes in themicrowave oven, and mechanical homogenization was repeated for anadditional 5 minutes (e.g., until the mixture was liquefied andhomogeneous).

Ground demineralized bone powder was prepared by impact fragmentation ofbone, followed by freeze-drying. The freeze-dried particles were sizedusing mesh sieves. Ground demineralized bone particles having a size inthe range of about 250 to 710 microns and demineralized to an averageweight percent residual calcium of 2±1% were used.

In order to prepare the tissue repair composition, the freeze-drieddemineralized bone powder was added to the heated, homogenized tendontissue until the final concentration of the bone in the tissue repaircomposition was about 30% by weight. Samples were sealed in sterilizedglass vials in 2 g aliquots.

Example 3 Preparation of Tissue Repair Compositions ContainingNon-Freeze-Dried Fascia

Fascia and bone from a human cadaver were procured and returned to theprocessing facility under sterile conditions. Donor histories, personaland medical, were obtained following accepted standards of the AmericanAssociation of Tissue Banks Microbiological tests were performedfollowing FDA guidelines for testing sterility of products.

The bone and fascia were cleaned of unwanted tissues and the bone wasfreeze-dried. Fresh, non-freeze-dried fascia was used. The fascia wascut into long strips and was mixed with water at a ratio of about 1:15by weight. The ingredients were heated to a temperature of 100° C. usinga microwave oven, and maintained at this temperature for 4 minutes.Water was added to the heated composition to replace the liquid lost toevaporation. The heated composition was transferred into a conventionalblender and mechanically homogenized (e.g., blended) for 5 minutes. Thehomogenized connective tissue was re-heated for an additional 4 minutesin the microwave oven, and mechanical homogenization was repeated for anadditional 5 minutes (e.g., until the mixture was liquefied andhomogeneous).

Ground demineralized bone powder was prepared by impact fragmentation ofbone, followed by freeze-drying. The freeze-dried particles were sizedusing mesh sieves. Ground demineralized bone particles having a size inthe range of about 250 to 710 microns and demineralized to an averageweight percent residual calcium of 2±1% were used.

To prepare a first tissue repair composition, the demineralized bonepowder was added to the homogenized, non-freeze-dried fascia until thefinal concentration of the bone was about 30% by weight. In a secondtissue repair composition, the demineralized bone powder was added to afinal concentration of about 65% by weight. Samples of the tissue repaircompositions were sealed in sterilized glass vials in 2 g aliquots.

Example 4 Preparation of a Tissue Repair Composition ContainingRehydrated Freeze-Dried Fascia and Rehydrated Freeze-Dried Bone

Fascia and bone from a human cadaver were procured and returned to theprocessing facility under sterile conditions. Donor histories, personaland medical, were obtained following accepted standards of the AmericanAssociation of Tissue Banks Microbiological tests were performedfollowing FDA guidelines for testing sterility of products.

The bone and fascia were cleaned of unwanted tissues and freeze-dried.The fascia was rehydrated prior to being used in making of thecomposition. The rehydrated fascia was cut into small (about ½ cm by ½cm) pieces (e.g., crude fragments). Isotonic saline in a volume (1 cm³of isotonic saline corresponds to about 1 g) approximating 20 times theweight of tissue, was added to a container containing the cut fascia.The ingredients were heated to a temperature of 100° C. using amicrowave oven, and maintained at this temperature for 4 minutes. Waterwas added to the heated composition to replace the liquid lost toevaporation. The heated composition was transferred into a conventionalblender and mechanically homogenized (e.g., blended) for 5 minutes. Thehomogenized connective tissue was re-heated for an additional 4 minutesin the microwave oven, and mechanical homogenization was repeated for anadditional 5 minutes (e.g., until the mixture was liquefied andhomogeneous).

The ground demineralized bone powder was prepared by impactfragmentation, followed by freeze-drying, and finally the particles weresized using mesh sieves. Ground demineralized bone particles having asize in the range of about 125 to 500 microns were used.

The freeze-dried demineralized bone particles were rehydrated prior tobeing combined with the homogenized fascia. The final concentration ofthe bone particles in the tissue repair composition was about 20 wt %after rehydration. The resulting tissue repair composition was a gel.The gel was packaged in 5 ml Luerlock syringes. The gel was easilyextruded from the syringe. A thirteen-gauge needle was attached to thesyringe, and the gel was easily extruded through the needle, as well.

Example 5 Preparation of a Molded Tissue Repair Composition ContainingFreeze-Dried Fascia

Fascia and bone from a human cadaver were procured and returned to theprocessing facility under sterile conditions. Donor histories, personaland medical, were obtained following accepted standards of the AmericanAssociation of Tissue Banks Microbiological tests were performedfollowing FDA guidelines for testing sterility of products.

The bone and fascia were cleaned of unwanted tissues and freeze-dried.The freeze-dried fascia was cut into small (about ½ cm by ½ cm) pieces(e.g., crude fragments). Isotonic saline in a volume (1 cm³ of isotonicsaline corresponds to about 1 g) approximating 20 times the weight oftissue, was added to a container containing the cut fascia. Theingredients were heated to a temperature of 100° C. using a microwaveoven, and maintained at this temperature for 4 minutes. Water was addedto the heated composition to replace the liquid lost to evaporation. Theheated composition was transferred into a conventional blender andmechanically homogenized (e.g., blended) for 5 minutes. The homogenizedconnective tissue was re-heated for an additional 4 minutes in themicrowave oven, and mechanical homogenization was repeated for anadditional 5 minutes (e.g., until the mixture was liquefied andhomogeneous).

Ground demineralized bone powder was prepared by impact fragmentation ofbone, followed by freeze-drying. The freeze-dried particles were sizedusing mesh sieves. Ground demineralized bone particles having a size inthe range of about 125 to 710 microns and demineralized to an averageweight percent residual calcium of 2±1% were used.

In order to prepare the tissue repair composition, the freeze-drieddemineralized bone powder was added to the homogenized fascia until thefinal concentration of the bone in the composition was about 40% byweight. The tissue repair composition was then placed into differentcontainers and molds, and freeze-dried using a two-day cycle asprescribed by the manufacturer of the freeze-drier. The freeze-dried,molded, tissue repair compositions demonstrated high mechanical strengthand maintained the shape of their mold. The cast tissue repaircomposition may be rehydrated using an isotonic solution to make itmalleable or may be cross-linked with a fixative such as glutaraldehyde,EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), or genapin to helpretain its solid, rigid, molded form to be used in applications where aspecific shape and mechanical strength would be desirable.

Example 6 Preparation of Tissue Repair Compositions ContainingFreeze-Dried Fascia

Fascia and bone from a human cadaver were procured and returned to theprocessing facility under sterile conditions. Donor histories, personaland medical, were obtained following accepted standards of the AmericanAssociation of Tissue Banks Microbiological tests were performedfollowing FDA guidelines for testing sterility of products. The bone andfascia were cleaned of unwanted tissues and freeze-dried.

Ground demineralized bone powder was prepared by impact fragmentation ofbone, followed by freeze-drying. The freeze-dried particles were sizedusing mesh sieves. Ground demineralized bone particles having a size inthe range of about 125 to 500 microns and demineralized to an averageweight percent residual calcium of 2±1% were used.

The small pieces of connective tissue and the saline solution werebrought to a temperature of 100° C. using a heating plate and themixture was heated at this temperature for an additional 5 minutes.Water was added to the mixture to replace the solution lost due toevaporation. The mixture was transferred into a conventional blender andmechanically homogenized at approximately 15,000 rpm (maximum shearspeed of the commercially available blender) for 5 minutes. The mixturewas again heated to a temperature of 100° C. using the heating plate,and maintained at this temperature for an additional 5 minutes. Theheated mixture was again blended for two, 2-minute pulses to produce thehomogenized fascia.

To prepare a first tissue repair composition, the sized, freeze-drieddemineralized bone powder was added to the homogenized fascia until thefinal concentration of the bone was about 30% by weight. In a secondtissue repair composition, the demineralized bone powder was added tothe homogenized fascia until the final concentration of the bone wasabout 50% by weight.

Example 7 Preparation of Tissue Repair Compositions ContainingFreeze-Dried Fascia

Fascia and bone from a human cadaver were procured and returned to theprocessing facility under sterile conditions. Donor histories, personaland medical, were obtained following accepted standards of the AmericanAssociation of Tissue Banks Microbiological tests were performedfollowing FDA guidelines for testing sterility of products.

The bone and fascia were cleaned of unwanted tissues and freeze-dried.The freeze-dried fascia was cut into small, ½ cm by ½ cm pieces (e.g.,crude fragments). Isotonic saline in a volume (1 cm³ of isotonic salinecorresponds to about 1 g) approximating 50 times the weight of thetissue to be processed was added to cut fascia. The fascia and salinewere brought to a temperature of 100° C. using a heating plate and washeated at this temperature for an additional 5 minutes. Water was addedto replace the solution lost due to evaporation. The heated material wastransferred into a blender and mechanically modified at 15,000 rpm(maximum shear speed of the commercially available blender) for 5minutes. The homogenized connective tissue was again heated to atemperature of 100° C. using the heating plate, and maintained at thistemperature for an additional 5 minutes. The heated homogenate wasblended for two, 2-minute pulses. The material was divided and placedinto centrifuge containers and spun at 1000 rcf for 5, 7, 9, and 10minutes, respectively. Water in the material separated into a distinctlayer after the centrifugation process. The volume of the water-layerwas proportional to the centrifugation time. The materials remainingafter removal of the water layer had different consistencies. To preparetissue repair compositions, the sized, freeze-dried demineralized bonepowder was added to the various homogenized materials until the finalconcentration of the bone was about 30% by weight. The viscosities ofthe tissue repair compositions correlated to the differing consistenciesof the homogenized connective tissue materials used in theirpreparation.

Example 8 Determination of New Bone Formation

Fascia lata and bone from a human cadaver were procured and returned tothe processing facility under sterile conditions. Donor histories,personal and medical, were obtained following accepted standards of theAmerican Association of Tissue Banks Microbiological tests wereperformed following FDA guidelines for testing sterility of products.

The bone and fascia were cleaned of unwanted tissues and freeze-dried.The freeze-dried fascia was cut into small (about ½ cm by ½ cm) pieces(e.g., crude fragments). Isotonic saline in a volume (1 cm³ of isotonicsaline corresponds to about 1 g) approximating 20 times the weight oftissue, was added to a container containing the cut fascia. Theingredients were heated to a temperature of 100° C. using a microwaveoven, and maintained at this temperature for 4 minutes. Water was addedto the heated composition to replace the liquid lost to evaporation. Theheated composition was transferred into a conventional blender andmechanically homogenized (e.g., blended) for 5 minutes. The homogenizedconnective tissue was re-heated for an additional 4 minutes in themicrowave oven, and mechanical homogenization was repeated for anadditional 5 minutes (e.g., until the mixture was liquefied andhomogeneous).

Ground demineralized bone powder was prepared by impact fragmentation ofbone, followed by freeze-drying. The freeze-dried particles were sizedusing mesh sieves. Ground demineralized bone particles having a size inthe range of about 250 to 710 microns and demineralized to an averageweight percent residual calcium of 2±1% were used.

Tissue repair compositions having a putty-like consistency were preparedby adding the sized, freeze-dried demineralized bone powder to thehomogenized fascia until the final concentration of the bone was about24%, 26%, 30%, and 50% by weight, respectively.

The prepared tissue repair compositions, a demineralized bone matrix(DBM) control (without homogenized connective tissue), and a homogenizedtissue sample without DBM were implanted heterotopically (e.g., intomuscle pouches) in the hind quarters of athymic (e.g., nude) mice. Eachimplant (other than the homogenized tissue-only sample) contained 20 mgof demineralized bone matrix. The amounts of materials implanted werevaried to always implant 20 mg of DBM i.e., 40 mg of the 50% DBMcomposition was implanted. In that the DBM constituted 50% by weight,and the homogenized connective tissue constituted 50% by weight, thetotal implant of 40 mg contained 20 mg of DBM. Thus, 85 mg, 77 mg, 68mg, and 40 mg of the 24 wt %, 26 wt %, 30 wt %, and 50 wt % tissuerepair compositions were implanted, respectively. Three mice with twoimplants per mouse were used for each of the four tissue repaircompositions, the DBM control, and the homogenized tissue-only sample(e.g., 18 mice in all and 36 implants).

After 28 days, the implants were explanted, and one explant from eachmouse was fixed. At least one histological section was cut from thecenter of each of these explants. Samples were fixed in 10% bufferedformalin. Standard dehydration, embedding and sectioning protocols wereused to produce light microscopy slides that were subsequently stainedwith hematoxylin and eosin. Using histomorphometric analysis, thepercent new bone formed was calculated as a cross-sectional area ofnewly formed bone (mm²) divided by the total cross-sectional area (mm²)for a representative microscopic view of a histology slide multiplied by100. Every other field of view with at least 50% bone content was usedas a representative view with about 10 representative views beinganalyzed per slide.

The demineralized bone that was implanted without homogenized connectivetissue (e.g., the control) produced about 9.6% new bone growth, and thehomogenized fascia alone (freeze dried material) produced about 3.8% newbone growths. The 24%, 26%, 30%, 50% demineralized bone to homogenizedconnective tissue compositions resulted in about 6.4%, 11.1%, 14.5%, and16.2% new bone growth, respectively.

Example 9 Use of Radioprotectants

To meet the current U.S. FDA regulatory standards and marketexpectations, a product must be sterile to a sterility assurance levelof 1×10⁻⁶ (SAL). It was found that tissue repair compositions comprisingHCT and bone fragments after gamma irradiation do not maintain theirmalleablity and tenaciousity after irradiation. The composition crumbledapart releasing the bone matrix.

In order to manufacture a tissue repair composition, in the form of aputty/paste, having desirable handling characteristics post-irradiationat dose designed to achieve a SAL of 1×10⁻⁶, it was thought that theaddition of one or more radioprotectants to the composition wouldproduce a product with the desirable handling properties because theradioprotectant will act as a free radical scavenger during theirradiation sterilization process and would protect the handlingcharacteristics of the putty/paste composition. An addition facet wasthat the radioprotectant must not negatively interfere with thebiological activity over time (shelf life).

Multiple radioprotectants (Table 1) were investigated over a range ofconcentrations and the handling characteristics of the resultantproducts were semi-quantitatively analyzed post-gamma irradiation. Thoseproducts that passed the semi-quantitative analysis went on to beimplanted into athymic nude mice as a bioassay to assessosteoinductivity. For example, 100 mM aminoguanidine was found to haveno negative impact on osteoinductivity and the handling characteristicsafter receiving a sterilizing dose of gamma irradiation.

The method to add the radioprotectant was consistent for eachradioprotectant investigated. The slurry was made as described inexample 1 (heating and blending cycles) and once completed, theradioprotectant of choice was added to the slurry in the form it wassupplied from the manufacturer (powder or liquid).

TABLE 1 Radioprotectants and Concentration Investigated RadioprotectantPassed Osteoinductivity Aminoguanidine Yes (CAS 1937-19-5) L-AscorbicAcid, salt Yes (CAS 14306-25-3) L-Ascorbic Acid 2- Yes phosphatesesquimagnesium salt (CAS 113170-55-1)

Example 10 Preparation of Tissue Repair Composition ContainingNon-Demineralized Bone and Fascia

Fascia lata and bone from a human cadaver were procured and returned tothe processing facility under sterile conditions. Donor histories,personal and medical, were obtained following accepted standards of theAmerican Association of Tissue Banks Microbiological tests wereperformed following FDA guidelines for testing sterility of products.

The bone and fascia were cleaned of unwanted tissues and freeze-dried.The freeze-dried fascia was cut into small (about 1.5 cm by 1.5 cm)pieces (e.g., crude fragments). Sterile water in a volume (1 cm³ ofisotonic saline corresponds to about 1 g) approximating 20 times theweight of tissue, was added to a container containing the cut fascia.The ingredients were heated to a temperature of 100 degree C. using ahot plate, and maintained at this temperature for about 5 minutes.Optionally, water was added to the heated composition to replace theliquid lost to evaporation. The heated composition was transferred intoa conventional blender and mechanically homogenized (e.g., blended) forabout 1 to 3 minutes. The homogenized connective tissue was re-heatedfor an additional 3 minutes on a hot plate, and mechanicalhomogenization was repeated for an additional 4 minutes (e.g., until themixture was liquefied and homogeneous).

Ground cortical (GC) bone powder was prepared by impact fragmentation ofbone, followed by freeze-drying. The freeze-dried particles were sizedusing mesh sieves. Ground cortical bone particles having a size in therange of about 250 to 1000 microns were used.

To prepare the tissue repair implant, the homogenized fascia was mixedwith the sized, freeze-dried ground cortical bone powder and glycerol,and freeze dried. The bone was about 15%, 30%, 50%, 60%, 85%, 95% byweight in the tissue repair composition. The tissue repair compositioncan be aliquoted to different volumes and optionally freeze-dried.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention. Other aspects, advantages, and modifications are within thescope of the following claims.

The invention claimed is:
 1. A carrier consisting essentially of one ormore homogenized connective tissues.
 2. A tissue repair compositioncomprising the carrier of claim 1 and a plurality of bone fragments. 3.The tissue repair composition of claim 2, wherein the bone fragments arenatural demineralized bone fragments, natural non-demineralized bonefragments, synthetic bone fragments, or a combination thereof.
 4. Thetissue repair composition of claim 3, wherein the tissue repaircomposition further comprises a bioactive factor.
 5. The tissue repaircomposition of claim 3, wherein the tissue repair composition furthercomprises a radioprotectant.
 6. The tissue repair composition of claim5, wherein the radioprotectant is aminoguanidine.
 7. A carriercomposition consisting essentially of the carrier of claim 1 and a watermiscible polar organic compound.
 8. The carrier composition of claim 7,wherein the water miscible polar organic compound is glycerol.
 9. Acomposition consisting essentially of the carrier composition of claim 8and a radioprotectant.
 10. The composition of claim 9, wherein theradioprotectant is aminoguanidine.
 11. A tissue repair compositioncomprising the carrier composition of claim 10 and a plurality of bonefragments.
 12. The tissue repair composition of claim 11, wherein thebone fragments are natural demineralized bone fragments, naturalnon-demineralized bone fragments, synthetic bone fragments, or acombination thereof.
 13. A kit comprising the carrier composition ofclaim 1 and bone fragments.
 14. A carrier composition consistingessentially of (i) a carrier consisting essentially of one or morehomogenized connective tissues and (ii) one or more compounds selectedfrom the group consisting of radioprotectants, water replacement agents,water miscible polar organic compounds, water, natural polymers,synthetic polymers, antibiotics, antiviral agents, and polysaccharides.15. A composition consisting essentially of the carrier composition ofclaim 14 and a bioactive factor.
 16. The composition of claim 14,wherein the radioprotectants are aminoguanidine, ascorbic acid, phyticacid, or a combination thereof.
 17. The composition of claim 14, whereinthe water miscible polar organic compounds are glycerol, sugar alcohols,sugars, alcohols, or lipids.
 18. A tissue repair composition comprisingthe carrier composition of claim 14 and a plurality of bone fragments.19. The tissue repair composition of claim 18, wherein the bonefragments are natural demineralized bone fragments, naturalnon-demineralized bone fragments, synthetic bone fragments, or acombination thereof.
 20. The tissue repair composition of claim 19,wherein the carrier composition comprises aminoguanidine.
 21. The tissuerepair composition of claim 20, wherein the tissue repair compositionfurther comprises a bioactive factor.